The growing production of beef has resulted in the increased volume of liquid residues generated during slaughtering and processing, known as slaughterhouse wastewater. In an attempt to reduce the organic content of this wastewater, blood has been separated from the water, generating slaughterhouse wastewater from green line slaughter and processing (SHWW in natura). The objective of this study was to determine the physical, chemical, and toxicological characteristics of SHWW in natura collected from a bovine slaughterhouse and locker. The results obtained from this characterization showed a high concentration of solids, total volatile acids, alkalinity, macro and micro nutrients, and organic matter (BOD5 20°C, COD, and DOC). The effluent presented acute toxicity to the test-organisms P. putida, E. coli, and D. similis, and chronic toxicity to the test-organisms C. silvestri, C. dubia, E. coli, P putida, and P. subcaptata. Based on the ecotoxicological tests, it was concluded that the compounds of the SHWW in natura were more toxic to microcrustaceans and algae than to bacteria.